The storage and mobilization of lipid are fundamental cellular processes that are conserved throughout metazoan evolution and are present in virtually all mammalian cells. Intracellular FFA (FFAi) are normally kept under tight control since excessive FFAi are toxic. Indeed, excessive accumulation of intracellular FFA and FFA metabolites leads to cellular dysfunction, termed 'lipotoxicity', which is thought to be a major means by which obesity contributes to diabetes and cardiovascular disease. Thus, a mechanistic understanding of how cells assimilate, mobilize and channel FFA is an important biological question with broad implications for health and disease. The uptake and mobilization of FFA within cells appear to have important temporal and spatial domains. Nonetheless, investigation of the when and where FFA metabolism occurs within live cells has not been possible because intracellular FFA levels cannot presently be imaged. Therefore, the aim of the R21 application is to develop and implement an optical sensor of FFAi having high spatial and temporal resolution for biological applications. This sensor is designed to be used as a general cytosolic sensor, or can be specifically targeted to proposed subcellular sites of FFA uptake, mobilization and oxidation. We anticipate that a validated sensor will have wide applications and will lead to new insights into biology of cellular lipid trafficking. PUBLIC HEALTH RELEVANCE: The storage and mobilization of lipid are fundamental cellular processes that are present in virtually all mammalian cells. Intracellular FFA (FFAi) are normally kept under tight control and since excessive FFAi are toxic and can contribute to obesity-related diabetes and cardiovascular disease. Thus, a mechanistic understanding of how cells assimilate, mobilize and channel FFA is an important biological question with broad implications for health and disease. Evidence indicates that the uptake and mobilization of FFA within cells has important temporal and spatial domains, however, analysis of these dynamics has not been difficult because intracellular FFA levels cannot presently be imaged. Therefore, the aim of the R21 application is to develop and implement an optical sensor of intracellular FFA with high spatial and temporal resolution for biological applications. This sensor can be used as a general cytosolic sensor, or can be specifically targeted to key subcellular sites of FFA uptake, mobilization and oxidation. We anticipate that a validated sensor will have wide applications and will lead to new insights into biology of cellular lipid trafficking.